Compositions and methods for detecting a nucleic acid sequence in a sample comprising a primer oligonucleotide with a 3′-terminal region comprising a 2′-modified nucleotide

ABSTRACT

The present invention features compositions and methods for quantifying detection of a target oligonucleotide in a sample in real time comprising one or more primer oligonucleotides comprising a 5′ nicking enzyme recognition site and a 3′-terminal region comprising a 2′-modified nucleotide. These methods are compatible with amplification of target oligonucleotides in a test sample, including biological samples, using a nicking amplification reaction.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/808,442, filed Nov. 9, 2017, now U.S. Pat. No. 10,077,467; which is acontinuation of U.S. patent application Ser. No. 15/438,330, filed Feb.21, 2017, abandoned; which is a continuation of U.S. patent applicationSer. No. 14/989,687, filed Jan. 6, 2016, now U.S. Pat. No. 9,631,231,which is a continuation of U.S. patent application Ser. No. 14/789,545,filed Jul. 1, 2015, now U.S. Pat. No. 9,322,053; which is a continuationof U.S. patent application Ser. No. 14/342,766, filed Mar. 4, 2014, nowU.S. Pat. No. 9,096,897, which is the U.S. national phase application,pursuant to 35 U.S.C. § 371, of PCT International Application Ser. No.PCT/US2013/035750, filed Apr. 9, 2013, designating the United States andpublished in English, which claims priority to and the benefit of U.S.Provisional Application No. 61/621,975, filed Apr. 9, 2012; each of theaforementioned applications are incorporated herein by reference intheir entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 28, 2018, isnamed 167665.010633_SL.txt and is 26,102 bytes in size.

BACKGROUND OF THE INVENTION

Nucleic acid amplification technologies have provided a means ofunderstanding complex biological processes, detection, identification,and quantification of pathogenic and non-pathogenic organisms, forensiccriminology analysis, disease association studies, and detection ofevents in genetically modified organisms, etc. The polymerase chainreaction (PCR) is a common thermal cycling dependent nucleic acidamplification technology used to amplify DNA consisting of cycles ofrepeated heating and cooling of the reaction for DNA melting andenzymatic replication of the DNA using a DNA polymerase. Real-Timequantitative PCR (qPCR) is a technique used to quantify the number ofcopies of a given nucleic acid sequence in a biological sample.Currently, qPCR utilizes the detection of reaction products in real-timethroughout the reaction and compares the amplification profile to theamplification of controls which contain a known quantity of nucleicacids at the beginning of each reaction (or a known relative ratio ofnucleic acids to the unknown tested nucleic acid). The results of thecontrols are used to construct standard curves, typically based on thelogarithmic portion of the standard reaction amplification curves. Thesevalues are used to interpolate the quantity of the unknowns based onwhere their amplification curves compared to the standard controlquantities.

In addition to PCR, non-thermal cycling dependent amplification systemsor isothermal nucleic acid amplification technologies exist including,without limitation: Nicking and Extension Amplification Reaction (NEAR),Rolling Circle Amplification (RCA), Helicase-Dependent Amplification(HDA), Loop-Mediated Amplification (LAMP), Strand DisplacementAmplification (SDA), Transcription-Mediated Amplification (TMA),Self-Sustained Sequence Replication (3SR), Nucleic Acid Sequence BasedAmplification (NASBA), Single Primer Isothermal Amplification (SPIA),Q-β Replicase System, and Recombinase Polymerase Amplification (RPA).

NEAR amplification has similarities to PCR thermocycling. Like PCR, NEARamplification employs oligonucleotide sequences which are complementaryto a target sequences referred to as primers in PCR and templates inNEAR. In addition, NEAR amplification of target sequences results in alogarithmic increase in the target sequence, just as it does in standardPCR. Unlike standard PCR, the NEAR reaction progresses isothermally. Instandard PCR, the temperature is increased to allow the two strands ofDNA to separate. In a NEAR reaction, the target nucleic acid sequence isnicked at specific nicking sites present in a test sample. Thepolymerase infiltrates the nick site and begins complementary strandsynthesis of the nicked target nucleotide sequence (the added exogenousDNA) along with displacement of the existing complimentary DNA strand.The strand displacement replication process obviates the need forincreased temperature. At this point, template/primer molecules annealto the displaced complementary sequence from the added exogenous DNA.The polymerase now extends from the 3′ end of the template, creating acomplementary strand to the previously displaced strand. The secondtemplate/primer oligonucleotide then anneals to the newly synthesizedcomplementary strand and extends making a duplex of DNA which includesthe nicking enzyme recognition sequence. This strand is then liable tobe nicked with subsequent strand displacement extension by thepolymerase, which leads to the production of a duplex of DNA which hasnick sites on either side of the original target DNA. Once this issynthesized, the molecule continues to be amplified exponentiallythrough replication of the displaced strands with new templatemolecules. In addition, amplification also proceeds linearly from eachproduct molecule through the repeated action of the nick translationsynthesis at the template introduced nick sites. The result is a veryrapid increase in target signal amplification; much more rapid than PCRthermocycling, with amplification results in less than ten minutes.

Quantification has been problematic, however. Optimal performance of areal-time NEAR system depends on the generation and amplification of aspecific product. NEAR systems are known to generate significant levelsof non-specific background products in addition to the specific productby the reaction enzymes. These background products can serve asamplifiable entities and their generation can out-compete the generationof specific product. While it is possible to design detection probesspecific to the desired target (and thus the specific product isdetectable in a complex background), significant levels of non-specificbackground products sequester reaction components that may haveotherwise been utilized for the amplification of the specific product.Thus, sequestration of reaction components due to non-specificbackground product generation results in a reaction that is suboptimal.This is particularly troublesome when the target nucleic acid isinitially in very low abundance and where a highly optimized reaction isrequired for reliable detection of the target. Also, a suboptimalreaction may not represent true quantification of a target nucleic acideven though it is detectable. It would be advantageous to generateoptimized NEAR reactions that eliminate the amplification ofnon-specific background products. Doing so would provide a reaction thatis suitable for quantification either by a standard curve based systemor relative quantification.

Also, it is common practice to evaluate NEAR reactions using massspectrometry. High levels of background products can obscure theinterpretation of mass spectrometry data. If, for instance, a reactioncontains background products, one or more products derived fromnon-specific amplification (from related yet dissimilar targets), andthe specific product, it would be challenging to identify thesematrix-derived products from the background products. Elimination ofbackground products leads to a clear determination of theperformance/specificity of the particular assay.

Additionally, high levels of background products can impede the optimalamplification of intentionally-duplexed or multiplexed reactions. Whilemultiple, differentially labeled detection probes are compatible withreal-time detection, there still exists the problem of reactantlimitations due to non-specific product formation. This is particularlytrue for duplex or multiplex reactions in that these reactions containmore than two templates/primers that can potentially form complexpopulations of background products. A NEAR reaction system thateliminates the amplification of background products also providesconditions for the detection of intentionally duplexed or multiplexedreactions in real time. It would be highly advantageous to provide ameans to eliminate amplifiable background products thus maximizing thepotential of generating specific products in NEAR reactions. It would bedesirable if a quantitative result could be provided by accuratelymonitoring the progress of the reaction in real-time.

SUMMARY OF THE INVENTION

As described below, the present invention features compositions andmethods for detection of a target oligonucleotide in a sample in realtime that reduces or eliminates the generation of background products,allowing for the quantification of the sample target oligonucleotide.These methods are compatible with target oligonucleotides amplifiedusing a NEAR reaction. The invention is based, at least in part, on thediscovery that specific products in single-plexed NEAR reactions can begenerated without the generation of background products. The reactioncompositions and methods provide for relative quantification of unknowntest samples, duplexed reactions, and multiplexed reactions, and thecreation of standard curves for absolute quantification of unknown testsamples.

In one aspect, the invention provides a method of quantifying a specificproduct in a nicking and extension amplification reaction, the methodinvolving: contacting a target nucleic acid molecule under substantiallyisothermal conditions with an exonuclease deficient polymerase, two ormore primer/template oligonucleotides, each of which specifically bindsto a complementary sequence on the target nucleic acid molecule, anicking enzyme, and a detectable polynucleotide probe, where each of theprimer/template oligonucleotides has one or more 2′ modified nucleotidesin the sequence complementary to the target nucleic acid molecule;generating amplicons having at least a portion of said target nucleicacid molecule; and detecting a signal specific for oligonucleotide probehybridization to the target nucleic acid molecule or amplicon thereof,where the signal indicates the quantity of the target nucleic acidmolecule present in the sample or an amplicon thereof.

In another aspect, the invention provides a method for detecting aplurality of distinct reaction products produced in the course of asingle reaction, the method involving: contacting a target nucleic acidmolecule under substantially isothermal conditions with an exonucleasedeficient polymerase, two or more primer/template oligonucleotides, eachof which specifically binds to a complementary sequence on the targetnucleic acid molecule, a nicking enzyme, and a detectable polynucleotideprobe, where each of the primer/template oligonucleotides has one ormore 2′ modified nucleotides in the sequence complementary to the targetnucleic acid molecule; generating amplicons having at least a portion ofsaid target nucleic acid molecule; and detecting a signal specific foroligonucleotide probe hybridization to the target nucleic acid moleculeor amplicon thereof, where the signal indicates the quantity of thetarget nucleic acid molecule present in the sample or an ampliconthereof.

In a particular aspect, the invention provides a method of quantifying aspecific product in a nicking and extension amplification reaction, themethod involving: contacting a target nucleic acid molecule undersubstantially isothermal conditions with an exonuclease deficientpolymerase, two primer/template oligonucleotides, each of whichspecifically binds to a complementary sequence on the target nucleicacid molecule, a nicking enzyme, and a detectable polynucleotide probe,where each of the primer/template oligonucleotides has at least about 5contiguous 2′-O-methyl modified nucleotides are positioned at oradjacent to the 3′ end of the sequence complementary to the targetnucleic acid molecule (e.g., the 3′ terminus of the oligonucleotide);generating amplicons having at least a portion of said target nucleicacid molecule; and detecting a signal specific for oligonucleotide probehybridization to the target nucleic acid molecule or amplicon thereof,where the signal indicates the quantity of the target nucleic acidmolecule present in the sample or an amplicon thereof.

In one aspect, the invention provides a method for monitoring in realtime a nicking and extension amplification reaction, the methodinvolving: contacting a test sample with an exonuclease deficientpolymerase, two or more primer/template oligonucleotides, each of whichspecifically binds to a complementary sequence on the target nucleicacid molecule, a nicking enzyme, and a detectable polynucleotide probe,where each of the primer/template oligonucleotides has one or more 2′modified nucleotides in the sequence complementary to the target nucleicacid molecule under substantially isothermal conditions; generatingamplicons having at least a portion of said target nucleic acidmolecule; and detecting a signal in real time, thereby quantitating ofthe target nucleic acid molecule(s).

In another aspect, the invention provides a method for monitoring inreal time a target nucleic acid molecule in a NEAR reaction, the methodinvolving: contacting a target nucleic acid molecule under substantiallyisothermal conditions with an exonuclease deficient polymerase, two ormore primer/template oligonucleotides, each of which specifically bindsto a complementary sequence on the target nucleic acid molecule, anicking enzyme, a heteroduplex specific nicking enzyme, and a detectablepolynucleotide probe, where each of the primer/template oligonucleotideshas one or more 2′ modified nucleotides in the sequence complementary tothe target nucleic acid molecule; generating amplicons having a targetsequence that binds the detectable oligonucleotide probe; and detectinga signal in real time, thereby quantitating the target nucleic acidmolecule.

In yet another aspect, the invention provides a method for monitoring inreal time a target nucleic acid molecule in a test sample, the methodinvolving: contacting a target nucleic acid molecule under substantiallyisothermal conditions with a polymerase, two or more primer/templateoligonucleotides, each of which specifically binds to a complementarysequence on the target nucleic acid molecule, a nicking enzyme, a repairenzyme or proof reading enzyme, and a detectable polynucleotide probe,where each of the primer/template oligonucleotides has one or more 2′modified nucleotides in the sequence complementary to the target nucleicacid molecule; generating amplicons having a target sequence that bindsthe detectable oligonucleotide probe; and detecting a signal in realtime, thereby quantitating the target nucleic acid molecule.

In still another aspect, the invention provides a kit for detecting atarget sequence in a NEAR reaction, the kit containing one or moreprimer/template oligonucleotides, which specifically binds to acomplementary sequence on the target nucleic acid molecule and has oneor more 2′ modified nucleotides in the sequence complementary to thetarget nucleic acid molecule, and directions for use of theprimer/template oligonucleotide in methods of the invention.

In one aspect, the invention provides an isolated oligonucleotidehaving, from 5′ to 3′, a first region, and a second region, where thefirst region has a nicking enzyme recognition sequence; where the secondregion has at least 9 or more nucleotides (e.g., 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or morecontiguous nucleotides) that specifically bind a complementary sequenceon a target nucleic acid molecule; and where the second region has oneor more 2′ modified nucleotides. In other embodiments, the isolatedoligonucleotide is one set forth in FIG. 1.

In various embodiments of the aspects delineated herein, theoligonucleotide (e.g., primer/template oligonucleotide, isolatedoligonucleotide) contains a modified nucleotide, including a 2′ modifiednucleotide. In various embodiments of any aspect delineated herein, the2′ modification is one or more of a 2′-O-methyl, 2′-methoxyethoxy,2′-fluoro, 2′-hydroxyl, 2′-allyl, 2′-O-[2-(methylamino)-2-oxoethyl],4′-thio, 4′-CH₂—O-2′-bridge, 4′-(CH₂)₂—O-2′-bridge, 2′-LNA, 2′-alkyl,and 2′-O-(N-methylcarbamate) or the modified nucleotide contains a baseanalog. In various embodiments of any aspect delineated herein, one ormore 2′ modified nucleotides are positioned at or adjacent to the 3′ endof the sequence complementary to the target nucleic acid molecule (e.g.,the 3′ terminus of the oligonucleotide). In other embodiments of anyaspect delineated herein, one or more 2′ modified nucleotides arepositioned at the 5′ end of the sequence complementary to the targetnucleic acid molecule. In various embodiments of any aspect delineatedherein, one or more 2′ modified nucleotides positioned at the 5′ end ofthe sequence complementary to the target nucleic acid molecule areseparated from the nick site by 1, 2, 3, 4, 5 or more unmodifiednucleotides. In various embodiments of any aspect delineated herein, twoor more 2′ modified nucleotides are contiguous (2, 3, 4, 5, or more). Inother embodiments of any aspect delineated herein, two or more 2′modified nucleotides are alternating with unmodified nucleotides. Invarious embodiments of any aspect delineated herein, the nicking enzymerecognition sequence is 5′-GAGTC-3′. In various embodiments of anyaspect delineated herein, 5 contiguous 2′-O-methyl modified nucleotidesare positioned at or adjacent to the 3′ end of the sequencecomplementary to the target nucleic acid molecule (e.g., the 3′ terminusof the oligonucleotide). In other embodiments of any aspect delineatedherein, 5 contiguous 2′-O-methyl modified nucleotides are positioned atthe 5′ end of the sequence complementary to the target nucleic acidmolecule. In other embodiments of any aspect delineated herein, 2 ormore 2′-O-methyl modified nucleotides alternating with unmodifiednucleotides are positioned at the 5′ end of the sequence complementaryto the target nucleic acid molecule (i.e. target specific region).

In various embodiments of any aspect delineated herein, the detectingstep does not detect an amplicon of a non-target molecule. In variousembodiments of any aspect delineated herein, the method is carried outin real time. In certain embodiments of any aspect delineated herein,the step of generating amplicons is carried out in real time (e.g., todetermine the quantity of target present in the reaction).

In various embodiments of any aspect delineated herein, the methodprovides a semi-quantitative and/or quantity threshold method ofdetermining the amount of nucleic acid molecule present in a biologicalsample prior to amplification. In various embodiments of any aspectdelineated herein, positioning one or more 2′ modified nucleotidesnearer the 5′ end of the sequence complementary to the target nucleicacid molecule increases the detection time of amplification. In variousembodiments of any aspect delineated herein, the method further involvesthe use of ratios of primer/template oligonucleotides to provideincreased resolution of reaction products resulting from differingquantities of starting target material. It has been found thatincreasing the ratio of primer/template oligo having one or more 2′modified nucleotides at the 3′ end of the recognition sequence toprimer/template oligo having one or more 2′ modified nucleotides at the5′ end of the recognition sequence contracted the signal curve andshifted the slope of the curve.

In various embodiments of any aspect delineated herein, the methodfurther involves the use of an amplification rate modifier to provideincreased resolution of reaction products resulting from differingquantities of starting target material. In various embodiments of anyaspect delineated herein, the target nucleic acid molecule is a DNA orRNA nucleic acid molecule. In various embodiments of any aspectdelineated herein, the detectable probe is SYBR green or a MolecularBeacon. In various embodiments of any aspect delineated herein, thedetectable probe is a non-amplifiable detectable polynucleotide probehaving at least about 10 nucleotides that are complementary to a targetsequence, a detectable moiety, and a polymerase-arresting molecule,where the polymerase-arresting molecule prevents a polymerase fromamplifying the probe under conditions that otherwise support polymeraseactivity.

In various embodiments of any aspect delineated herein, the test samplecontains a pathogen. In various embodiments of any aspect delineatedherein, the pathogen is a virus, bacteria, yeast or fungus. In variousembodiments of any aspect delineated herein, the test sample is abiological sample. In various embodiments of any aspect delineatedherein, the biological sample is a cell, tissue sample, or biologicalfluid (e.g., urine, semen, vaginal secretion, or stool). In variousembodiments of any aspect delineated herein, the test sample is anenvironmental sample.

The invention provides compositions and methods for detecting a targetnucleic acid molecule amplified using a NEAR reaction. Compositions andarticles defined by the invention were isolated or otherwisemanufactured in connection with the examples provided below. Otherfeatures and advantages of the invention will be apparent from thedetailed description, and from the claims.

Definitions

In this disclosure, “comprises,” “comprising,” “containing” and “having”and the like can have the meaning ascribed to them in U.S. patent lawand can mean “includes,” “including,” and the like; “consistingessentially of” or “consists essentially” likewise has the meaningascribed in U.S. patent law and the term is open-ended, allowing for thepresence of more than that which is recited so long as basic or novelcharacteristics of that which is recited is not changed by the presenceof more than that which is recited, but excludes prior art embodiments.

By “polymerase-arresting molecule” is meant a moiety associated with apolynucleotide template/primer that prevents or significantly reducesthe progression of a polymerase on the polynucleotide template.Preferably, the moiety is incorporated into the polynucleotide. In onepreferred embodiment, the moiety prevents the polymerase fromprogressing on the template.

By “polymerase extension” is meant the forward progression of apolymerase from a accessible 3′-hydroxyl group that incorporatesincoming monomers complementary to their opposing nucleotides on atemplate polynucleotide strand.

By “exonuclease deficient polymerase” is meant a DNA-dependent DNApolymerase and/or RNA-dependent DNA polymerase that is devoid of a 5′-3′exonuclease activity or that has virtually undetectable levels of suchactivity.

By “nucleotide adduct” is meant a moiety that is bound covalently orotherwise fixed to a standard nucleotide base.

As used herein, the term “detectable polynucleotide probe” refers toany, at least partially single stranded, polynucleotide labeled with adetectable moiety with a sequence region complementary to at least onestrand of the target sequence, which releases a detectable signal fromthe detectable moiety upon binding to the target sequence, whereassignal generation by that detectable moiety does depend on cleavage ofthe detectable polynucleotuide probe by a non-specific 5′-3′ exonucleaseactivity. An example of a “detectable polynucleotide probe” as usedherein is, but is not limited to, a fluorescent molecular beacon probeas described in prior art.

As used herein, the term “nucleic acid” refers to deoxyribonucleotides,ribonucleotides, or modified nucleotides, and polymers thereof insingle- or double-stranded form. The term encompasses nucleic acidscontaining known nucleotide analogs or modified backbone residues orlinkages, which are synthetic, naturally occurring, and non-naturallyoccurring, which have similar binding properties as the referencenucleic acid, and which are metabolized in a manner similar to thereference nucleotides. Examples of such analogs include, withoutlimitation, 2′ modified ribonucleotides (e.g., 2′-O-methylribonucleotides, 2′-F nucleotides).

As used herein, “modified nucleotide” refers to a nucleotide that hasone or more modifications to the nucleoside, the nucleobase, pentosering, or phosphate group. For example, modified nucleotides excluderibonucleotides containing adenosine monophosphate, guanosinemonophosphate, uridine monophosphate, and cytidine monophosphate anddeoxyribonucleotides containing deoxyadenosine monophosphate,deoxyguanosine monophosphate, deoxythymidine monophosphate, anddeoxycytidine monophosphate. Modifications include those naturallyoccurring that result from modification by enzymes that modifynucleotides, such as methyltransferases. Modified nucleotides alsoinclude synthetic or non-naturally occurring nucleotides. Synthetic ornon-naturally occurring modifications in nucleotides include those with2′ modifications, e.g., 2′-alkyl, such as 2′-O-methyl and2′-methoxyethoxy, 2′-fluoro, 2′-hydroxyl (RNA), 2′-allyl,2′-O-[2-(methylamino)-2-oxoethyl], 4′-thio, 4′-CH₂—O-2′-bridge,4′-(CH₂)₂—O-2′-bridge, 2′-LNA, and 2′-O-(N-methylcarbamate) or thosecomprising base analogs.

By “base substitution” is meant a substituent of a nucleobase polymerthat does not cause significant disruption of the hybridization betweencomplementary nucleotide strands.

By “specific product” is meant a polynucleotide product resulting fromthe hybridization of template oligonucleotides to a complementary targetsequence and subsequent polymerase mediated extension of the targetsequence.

By “nicking and extension amplification reaction” is meant alternatingcycles of nicking and extension leading to amplification of apolynucleotide of interest.

By “substantially isothermal condition” is meant at a single temperatureor within a narrow range of temperatures that does not varysignificantly. In one embodiment, a reaction carried out undersubstantially isothermal conditions is carried out at a temperature thatvaries by only about 1-5° C. (e.g., varying by 1, 2, 3, 4, or 5degrees). In another embodiment, the reaction is carried out at a singletemperature within the operating parameters of the instrument utilized.

By “nicking enzyme” is meant a polypeptide capable of recognizing andbinding to a specific structure in double stranded nucleic acidmolecules and breaking a phosphodiester bond between adjoiningnucleotides on a single strand upon binding to its recognized specificstructure, thereby creating a free 3′-hydroxyl group on the terminalnucleotide upstream of the nick site that can be extended by aexonuclease deficient polymerase.

By “nick site” is meant the position of a “broken” phosphodiester bondin one strand of a double stranded nucleic acid molecule hydrolyzed by anicking enzyme.

By “amplicon” is meant a polynucleotide or a multitude ofpolynucleotides generated during the amplification of a polynucleotideof interest. In one example, an amplicon is generated during apolymerase chain reaction.

By “semi-quantitative” is meant providing an estimate of relativequantity based on an internal control.

By “quantity threshold method” is meant providing an estimate ofquantity based on either exceeding or not exceeding in quantity acomparative standard.

By “amplification rate modifiers” is meant an agent capable of affectingeither the rate of polymerase extension or the rate of single strandnicking by the nicking enzyme, or both.

By “monitoring a reaction” is meant detecting the progress of areaction. In one embodiment, monitoring reaction progression involvesdetecting polymerase extension and/or detecting a complete NEARreaction.

“Detect” refers to identifying the presence, absence or amount of theanalyte to be detected.

By “detectable moiety” is meant a composition that when linked to amolecule of interest renders the latter detectable, via spectroscopic,photochemical, biochemical, immunochemical, or chemical means. Forexample, useful labels include radioactive isotopes, magnetic beads,metallic beads, colloidal particles, fluorescent dyes, electron-densereagents, enzymes (for example, as commonly used in an ELISA), biotin,digoxigenin, or haptens.

By “fragment” is meant a portion of a nucleic acid molecule. Thisportion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, or 90% of the entire length of the reference nucleic acidmolecule or polypeptide. A fragment may contain 5, 10, 15, 20, 30, 40,50, 60, 70, 80, 90, or 100 nucleotides.

“Hybridization” means hydrogen bonding, which may be Watson-Crick,Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementarynucleobases. For example, adenine and thymine are complementarynucleobases that pair through the formation of hydrogen bonds.

By “isolated polynucleotide” is meant a nucleic acid (e.g., a DNA) thatis free of the genes which, in the naturally-occurring genome of theorganism from which the nucleic acid molecule of the invention isderived, flank the gene. The term therefore includes, for example, arecombinant DNA that is incorporated into a vector; into an autonomouslyreplicating plasmid or virus; or into the genomic DNA of a prokaryote oreukaryote; or that exists as a separate molecule (for example, a cDNA ora genomic or cDNA fragment produced by PCR or restriction endonucleasedigestion) independent of other sequences. In addition, the termincludes an RNA molecule that is transcribed from a DNA molecule, aswell as a recombinant DNA that is part of a hybrid gene encodingadditional polypeptide sequence.

The terms “isolated,” “purified,” or “biologically pure” refer tomaterial that is free to varying degrees from components which normallyaccompany it as found in its native state. “Isolate” denotes a degree ofseparation from original source or surroundings. “Purify” denotes adegree of separation that is higher than isolation. A “purified” or“biologically pure” protein is sufficiently free of other materials suchthat any impurities do not materially affect the biological propertiesof the protein or cause other adverse consequences. That is, a nucleicacid or peptide of this invention is purified if it is substantiallyfree of cellular material, viral material, or culture medium whenproduced by recombinant DNA techniques, or chemical precursors or otherchemicals when chemically synthesized. Purity and homogeneity aretypically determined using analytical chemistry techniques, for example,polyacrylamide gel electrophoresis or high performance liquidchromatography. The term “purified” can denote that a nucleic acid orprotein gives rise to essentially one band in an electrophoretic gel.For a protein that can be subjected to modifications, for example,phosphorylation or glycosylation, different modifications may give riseto different isolated proteins, which can be separately purified.

As used herein, “obtaining” as in “obtaining an agent” includessynthesizing, purchasing, or otherwise acquiring the agent.

By “reference” is meant a standard or control condition. As is apparentto one skilled in the art, an appropriate reference is where an elementis changed in order to determine the effect of the element.

By “hybridize” is meant pair to form a double-stranded molecule betweencomplementary polynucleotide sequences (e.g., a gene described herein),or portions thereof, under various conditions of stringency. (See, e.g.,Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A.R. (1987) Methods Enzymol. 152:507).

By “subject” is meant a mammal, including, but not limited to, a humanor non-human mammal, such as a bovine, equine, canine, ovine, or feline.

By “target nucleic acid molecule” is meant a polynucleotide to beanalyzed. Such polynucleotide may be a sense or antisense strand of thetarget sequence. The term “target nucleic acid molecule” also refers toamplicons of the original target sequence.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive. Unless specifically stated orobvious from context, as used herein, the terms “a”, “an”, and “the” areunderstood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear fromcontext, all numerical values provided herein are modified by the termabout.

The recitation of a listing of chemical groups in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable or aspect herein includes that embodiment as any singleembodiment or in combination with any other embodiments or portionsthereof.

Any compositions or methods provided herein can be combined with one ormore of any of the other compositions and methods provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts exemplary polymerase-arresting entity structures.Black=Stabilizer Sequence, Blue=Nicking Enzyme Recognition Sequence,Green=Nicking Enzyme Spacer Sequence, Red=Target-Specific RecognitionSequence, A=Adenine, T=Thymine, G=Guanine, C=Cytosine, U=Uracil,mX=2′-O-methyl RNA base. Underlined base(s) delineates the modifiedsequence segment. FIG. 1 discloses SEQ ID NOS 1-57, 50, 42, 33, 23, and12, respectively, in order of appearance.

FIGS. 2A-2C depicts evaluation of dynamic range synthetic long-mer toClavibacter michiganensis sepidonicus (Cms) target DNA. Exemplaryresults from the titration of Cms synthetic “long-mer” target anddetection via a fluorobeacon are shown. NEAR assay for Cms performedwithout input target nucleic acid is indicated as No Target Control(NTC). FIG. 2A is a graph showing that the signal in the No TargetControls (NTC's) was suppressed in the 2′-O-methyl modifiedprimer/template-containing reactions. FIG. 2B is a graph showing thatthe Standard Curve displayed a wide dynamic range using the 2′-O-methyltemplate reactions. FIG. 2C is a comparison of exemplary mass spectradata showing the elimination of non-specific amplification products inNo Target Controls (NTC) of the Cms assay system using 2′-O-methylmodified primers/templates (right panel), as compared to unmodifiedprimers/templates (left panel). To determine the effect of the2′-O-methyl modified primers/templates on the generation of backgroundproducts, samples (10 μl) of No Target Control reactions depicted inFIG. 2A were analyzed via HPLC/Mass Spectrometry. Mass spectra dataclearly demonstrate in the presence of 2′-O-methyl modifiedprimers/templates the presence of only the expected molecular specieswas detected and complex background products generated in the presenceof unmodified primers/templates were eliminated.

FIG. 3 depicts 2′-O-methyl modification of templates/primers eliminatedbackground signal in NEAR assay using SYBR Green detection. Exemplaryamplification data using 2′-O-methyl modified primers/templates and theelimination of non-specific amplification products are shown. FIG. 3A isa graph showing that significant signal was observed in the No TargetControls (NTC's), indicating background product generation in theabsence of target DNA. FIG. 3B is a graph showing that in the2′-O-methyl modified template-containing reactions signal in the NoTarget Controls (NTC's) was suppressed.

FIG. 4 depicts 2′-O-methyl modification of templates/primers eliminatedbackground signal in NEAR assay using Molecular Beacon detection.Exemplary amplification data using 2′-O-methyl modifiedprimers/templates and the elimination of non-specific amplificationproducts are shown. FIG. 4A is a graph showing that significant signalwas observed in the No Target Controls (NTC's), indicating backgroundproduct generation in the absence of target DNA. FIG. 4B is a graphshowing that in the 2′-O-methyl modified template-containing reactionssignal in the No Target Controls (NTC's) was suppressed.

FIG. 5 depicts exemplary polymerase arresting entities using 2′-O-methylmodified primers/templates or ratios of 2′-O-methyl modifiedprimers/templates can be used to manipulate both the time-to-detectionand the efficiency of the reaction, thus ‘tuning’ the reactions.Schematic representations of exemplary 2′-O-methyl modifiedtemplates/primers for the tuning of a specific reaction are shown,including a primer/template having a block of five 2′-O-methylnucleotides at the 3′ end (“Terminal” template; left) and aprimer/template having a block of five 2′-O-methyl nucleotides startingat the 3^(rd) nucleotide after the nick site (“Nick+2” template”;right). Each tuning condition comprises specific ratios of forward andreverse templates with each set of templates having varying structures.

FIG. 6 depicts amplification plots demonstrating the utility of2′-O-methyl modification of templates/primers for the ‘tuning’ of aspecific reaction. Exemplary amplification data using 2′-O-methylmodified primers/templates are shown. All of the reactions (induplicate) contained 10,000 genome equivalents of Cms DNA. Each tuningcondition represents specific ratios of forward and reverse templateswith each set of templates having varying structures. The red circlesdemonstrate a shift in the time-to-detection for each tuning condition.Additionally, the log phase of each condition was contracted and theslope of the curve was shifted.

FIG. 7 depicts the design of two primer/template sets (TS3 & TS6) usedin a NEAR assay to amplify a fragment of the corn ADH1 gene. The targetspecific region in the sequences of TS3 & TS6 primer/template sets aresignificantly longer (15-17 bases) than in primer/template sets oftypical NEAR assays (9 to 12 bases) in the prior art. In the TS3primer/template set the block of 5 consecutive 2′-O-methyl modifiednucleotides adjacent to the 3′-terminus is preceded by an upstreamregion of 2′-O-methyl modified nucleotides alternating with unmodifiednucleotides starting with a 2′-O-methyl modified nucleotide 5 or 4nucleotides downstream from the nick site, respectively. In contrast toTS3, there are only five 2′-O-methyl modified nucleotides in each of theprimers/templates of the TS6 set, which form a block of consecutivenucleotides adjacent to the unmodified 3′-terminal nucleotide. FIG. 7discloses SEQ ID NOS 58-61, respectively, in order of appearance.

FIG. 8 shows amplification plots of the ADH1 assay using two sets ofprimers/templates (TS3 & TS6) recorded in the SYBRGreen dye detectionchannel.

FIG. 9 shows the amplification plots of the same assay reactionsrecorded in the ROX channel.

FIG. 10 depicts the amplification plots of the NTC ADH1 assay reactionsrecorded. Comparing the results shown in FIGS. 8 and 9 it becomesevident that only the primer/template set TS3 produces the ADH1-specificamplicon, while the signal generated by set TS6 is mostly based onnon-specific amplification of background products detected only bySYBRgreen.

DETAILED DESCRIPTION OF THE INVENTION

The invention features compositions and methods that are useful for thequantification of a target nucleic acid molecule in an isothermalreaction. In particular embodiments, the invention provides compositionsand methods for the quantification of a target nucleic acid molecule ina NEAR reaction (e.g., in real time).

The invention is based, at least in part, on the surprising discoverythat primer-template oligonucleotides comprising a 2′ modified (e.g.,2′-O-methyl, 2′-Fluoro) nucleotide reduces or eliminates illegitimateamplification by 5′-3′exonuclease deficient derivatives of Bst DNApolymerase I.

NEAR Reaction.

The NEAR reaction has been used as an endpoint reaction that providesfor the non-quantitative detection of target oligonucleotides. Theconventional NEAR assay comprises (1) a target nucleic acid molecule;(2) two oligonucleotide molecules that are analagous to the primermolecules of PCR; termed “template-primers” comprising some number ofoligonucleotides that are complementary to the target nucleic acidmolecule and a site that can be cleaved by a nicking enzyme; (3) dNTPs;(4) a strand displacing, 5′-3′-exonuclease deficient polymerase; and (5)a nicking enzyme. Current methods for quantifying the NEAR reaction,particularly in real time, are inadequate due in part to theillegitimate amplification of non-target molecules present in a samplethat can obscure detection of target sequences in a conventional NEARreaction. For example, there is a consistent undesirable amplificationin NEAR reactions that results in a detectable signal in the absence ofa target molecule or with signals that do not accurately reflect theamount of target nucleic acid molecule present in the reaction. Althoughthis provides for detection of an endpoint product, it fails to providefor real time monitoring of the reaction.

The present invention provides modified primer/template oligonucleotidesthat overcome the problem of accurately quantitating a target nucleicacid molecule in a NEAR reaction. It is particularly useful forquantitating a target nucleic acid molecule in a NEAR reaction in realtime. The invention is based, at least in part, on the discovery thatprimer-template oligonucleotides comprising a 2′ modified (e.g.,2′-O-methyl, 2′-Fluoro) reduces or eliminates illegitimate amplificationwithout preventing the extension of those modified primer-templates inorder to amplifiy the specific product. The primer/templateoligonucleotides of the invention are useful in NEAR reactionscomprising one or more of the aforementioned NEAR components.

In other embodiments, the invention provides for primer-templateoligonucleotides comprising a 2′ modified (e.g., 2′-O-methyl, 2′-Fluoro)that is positioned at or adjacent to the 3′ terminus of theprimer-template. Surprisingly, 2′-O-methyl nucleotides positioned in the3′-terminal region of a primer-template not only do comprise effectivepriming substrates for 5′-3′-exonuclease deficient derivatives of BstDNA polymerase I in isothermal DNA amplification reactions, but use ofsuch modified primer-templates completely suppresses nonspecificprimer-dimer amplification. This is particularly surprising becauseconventional thinking in the field of isothermal DNA amplificationteaches that modified nucleotides (e.g., 2′-O-methyl ribonucleotides,unmodified ribonucleotides) could only be introduced at the 5′-terminalregion of the primer/template away the 3′-terminus, because placement of2′-O-methoyl—as well as ribonucleotides within 6 nuleotides from the3′-terminus of a primer had been demonstrated to inhibit primerextension by DNA polymerases (include as references the patentapplication from Amersham and the patent from Qiagen). 5′-3′ exonucleasedeficient derivatives of Bst DNA polymerase I used in NEAR and otherisothermal amplification technologies (LAMP) belongs to polA-typebacterial DNA polymerases involved in low synthesis fidelity DNA repairprocesses. In contrast, high fidelity genome replication in bacteria iscatalyzed by DNAE- & POLC type DNA polymerase III holoenzymes, whichutilize exclusively RNA primers to initiate DNA replication. In thepublished prior art the discrimination between RNA and DNA primers wasthought to be one mechanism for preventing the interference of higherror rate DNA polymerase I enzymes with high fidelity genomereplication. In this context the surprising discovery that derivativesof Bst DNA polymerase I can efficiently utilize 2′-modifiedribonucleotides as primers for DNA synthesis is remarkable andcounterintuitive.

Primer-Template Design

Exemplary polymerase-arresting entity structures from 5′ to 3′ comprisea stabilizer sequence, nicking enzyme recognition sequence, nickingenzyme spacer sequence, and target specific recognition sequence, thetarget specific recognition sequence comprising one or more 2′ modifiednucleotides (e.g., 2′-O-methyl, 2′-methoxyethoxy, 2′-fluoro, 2′-allyl,2′-O-[2-(methylamino)-2-oxoethyl], 2′-hydroxyl (RNA), 4′-thio,4′-CH₂—O-2′-bridge, 4′-(CH₂)₂—O-2′-bridge, 2′-LNA, and2′-O-(N-methylcarbamate)). Without being bound to theory, it ishypothesized that incorporating one or more 2′ modified nucleotides inthe recognition regions renders those modified regions unsuitable toserve as template for polymerase extension in nonspecific intermolecularand/or intramolecular complexes formed by interactions ofprimers/templates (e.g., primer-dimer formation), and, thereby, reducesor eliminates the background signal in isothermal amplification. The 2′modified nucleotide preferably has a base that base pairs with thetarget sequence. In particular embodiments, two or more 2′ modifiednucleotides (e.g., 2, 3, 4, 5 or more 2′ modified nucleotides) in thetarget specific recognition region are contiguous (e.g., a block ofmodified nucleotides). In some embodiments, the block of 2′ modifiednucleotides is positioned at the 3′ end of the target specificrecognition region. In other embodiments, the block of 2′ modifiednucleotides is positioned at the 5′ end of the target specificrecognition region. When the block of 2′ modified nucleotides ispositioned at the 5′ end of the target specific recognition region, the2′ modified nucleotides may be separated from the nick site by one ormore non-modified nucleotides (e.g., 2, 3, 4, 5 or more 2′ unmodifiednucleotides). Applicants have found that positioning of one or more 2′modified nucleotides or of a block of 2′ modified nucleotides alters thekinetics of amplification. When the one or more 2′ modified nucleotidesor block of 2′ modified nucleotides are positioned at or near the 5′ endof the recognition region or proximal to the nick site, real-timeamplification reactions showed decreased time to detection.Additionally, the signal curve was contracted and the slope of the curveshifted. The applicants have also found that in recognition regionsexceeding 12 nucleotides in length a single block of 5 consecutive2′-modified nucleotide is not sufficient to suppress nonspecificamplification and therefore the entire recognition region up to 4 or 5nucleotides downstream from the nick site must be substituted by2′-modified nucleotides alternating with unmodified nucleotides.

In a related embodiment, ratios of a primer/template oligo having one ormore 2′ modified nucleotides can be used to alter the time-to-detectionand/or the efficiency of the reaction for the ‘tuning’ of reactions,resulting in a predictable control over amplification kinetics.Increasing the ratio of primer/template oligo having one or more 2′modified nucleotides at the 3′-end of the recognition sequence toprimer/template oligo having one or more 2′ modified nucleotides at the5′ end of the recognition sequence contracted the signal curve andshifted the slope of the curve. It is advantageous to be able to “tune”a reaction providing a means to manipulate both the time-to-detection aswell as the efficiency of the reaction. Relative quantification using aninternal control requires that two important conditions be met. First,it is beneficial to be able to modify a reaction's time-to-detectioncreating a non-competitive reaction condition. Thus, by affecting thecontrol reaction to be detectable at a later time-point (relative to thetarget of interest) the control reaction does not out-compete thespecific target of interest even when the target of interest is in lowinitial abundance. Second, to ensure a true relative abundancecalculation, it is required that the control and specific targetreactions have matched efficiencies. By controlling the efficiency ofeach reaction using a “tuning” condition enables reactions to be matchedallowing for satisfactory relative quantification calculations. Tuningthe reactions can be used to match efficiencies of target nucleic acidamplification and reference nucleic amplification (e.g., internalstandard) in quantitative PCR (qPCR). Additionally, amplification curvesof the target nucleic acid and the internal standard may be altered sotime of detection of their amplification products are separated, whileproviding the same efficiency for target nucleic acid amplification andinternal standard amplification. Through the use of specificcombinations and ratios of oligonucleotide structures within a reactionit is possible to create conditions which enable tuned reactionperformance.

In various embodiments, primer/template pairs are constructed with astem and loop configuration. The 5′ end of the primer/templateoligonucleotide comprises a self-complementary region that forms atleast part of the stem. In some embodiments of the invention the stemfurther encompasses at least a portion or all of the nicking enzymerecognition sequence. In other various embodiments of the invention thenicking enzyme recognition sequence in the primers-templates is not partof the double stranded stem structure, but resides within the mostlysingle stranded loop. This nicking enzyme recognition site is linked atthe 3′ end to a secondary-structure-free site comprising a nicking sitethat is linked at the 3′ end to a sequence that is complementary to atarget sequence. If desired, the sequence that is complementary to thetarget sequence may comprise a secondary structure or may be free ofsecondary structure. The presence of absence or secondary structure,which may comprise a self-complementary region, will be determined tooptimize the particular NEAR assay.

In one embodiment, the methods of the invention provide a NEAR reactionthat comprises the standard NEAR components, but also comprises anenzyme capable of nicking a RNA nucleotide when present in aheteroduplex with a complementary DNA strand. In one example, thecleaved RNA nucleotide will be present in a string of 4-15 non-cleavableRNA nucleotides (i.e. O-2-Me-RNAs) toward the 5′ end of the targetcomplementary region of the PTO, and the 3′ end of the templateoligonucleotide will have a 3′ terminal ‘cap’. Only upon complete properhybridization of the template oligonucleotide, with the heteroduplexcleaving molecule (i.e. RNase H) will be able to cleave the RNA base,creating a 3′ end for the nick translation enzyme to extend from; andallowing the NEAR reaction to progress to completion. Aberrant templatebinding (primer dimers, partial non-target hybridization, etc) will notlead to the RNA-DNA heteroduplex to form; and thus prevent theprogression of the NEAR reaction. These templates will only be amplifiedafter binding to a complementary nucleotide sequence through the removalof the 3′ polymerase extension ‘cap’. This will lead to an increasedlevel of specificity and sensitivity of the NEAR reaction.

The template oligonucleotides of the invention are included in a NEARreaction that comprises (1) a target nucleic acid molecule; (2) twotemplate oligonucleotide molecules comprising some number ofoligonucleotides that are complementary to the target nucleic acidmolecule and a site that can be cleaved by a nicking enzyme andcomprised of 4-15 RNA nucleotides, one of which is RNase liable; (3)dNTPs; (4) a strand displacing polymerase; (5) a nicking enzyme; and (6)a DNA-RNA heteroduplex RNA nicking enzyme and a 3′ terminal polymeraseextension cap. Accordingly, the invention provides a method of usingthese components to quantitate a target nucleic acid molecule.

The method involves contacting a target nucleic acid molecule undersubstantially isothermal conditions with a polymerase, two templateoligonucleotides, each of which specifically binds to a complementarysequence on the target nucleotide molecule, a nicking enzyme, and aDNA-RNA heteroduplex nicking enzyme (e.g. RNase H) with a 3′ terminalpolymerase extension cap; generating a detectable amplicon thatcomprises at least a portion of a template oligonucleotide that binds atarget sequence.

Target Nucleic Acid Molecules

Methods and compositions of the invention are useful for theidentification of a target nucleic acid molecule in a test sample. Thetarget sequences is amplified from virtually any samples that comprisesa target nucleic acid molecule, including but not limited to samplescomprising fungi, spores, viruses, or cells (e.g., prokaryotes,eukaryotes). In specific embodiments, compositions and methods of theinvention detect Clavibacter michiganensis subsp. michiganensis,Clavibacter michiganensis subsp. sepedonicus, Pseudomonas syringae pvTomato, Xanthomonas campestris pv Vesicatoria, Alternaria spp,Cladosporium spp, Fusarium oxysporum, Verticilium dahlia, Pseudomonascurrugata, Erwina carotovora, and Ralstonia solanacearum. Exemplary testsamples include body fluids (e.g. blood, serum, plasma, amniotic fluid,sputum, urine, cerebrospinal fluid, lymph, tear fluid, feces, or gastricfluid), tissue extracts, culture media (e.g., a liquid in which a cell,such as a pathogen cell, has been grown), environmental samples,agricultural products or other foodstuffs, and their extracts, DNAidentification tags. If desired, the sample is purified prior toinclusion in a NEAR reaction using any standard method typically usedfor isolating a nucleic acid molecule from a biological sample.

In one embodiment, primer/template oligonucleotides amplify a targetnucleic acid of a pathogen to detect the presence of a pathogen in asample. Exemplary pathogens include fungi, bacteria, viruses and yeast.Such pathogens may be detected by identifying a nucleic acid moleculeencoding a pathogen protein, such as a toxin, in a test sample.Exemplary toxins include, but are not limited to aflatoxin, choleratoxin, diphtheria toxin, Salmonella toxin, Shiga toxin, Clostridiumbotulinum toxin, endotoxin, and mycotoxin. For environmentalapplications, test samples may include water, liquid extracts of airfilters, soil samples, building materials (e.g., drywall, ceiling tiles,wall board, fabrics, wall paper, and floor coverings), environmentalswabs, or any other sample.

In one embodiment disclosed herein, primer/template oligonucleotidesamplify a target nucleic acid of plant used as an internal control inmolecular breeding experiments geared towards improving, for example,the plant's resistance to drought, the plant's resistance to herbicides,to predation by harmful insects. One example of such an internal controltarget nucleic reduced to praxis herein is the ADH1 gene(alcoholdehydrogenase 1) from corn.

Target nucleic acid molecules include double-stranded andsingle-stranded nucleic acid molecules (e.g., DNA, RNA, and othernucleobase polymers known in the art capable of hybridizing with anucleic acid molecule described herein). RNA molecules suitable fordetection with a detectable oligonucleotide probe or detectableprimer/template oligonucleotide of the invention include, but are notlimited to, double-stranded and single-stranded RNA molecules thatcomprise a target sequence (e.g., messenger RNA, viral RNA, ribosomalRNA, transfer RNA, microRNA and microRNA precursors, and siRNAs or otherRNAs described herein or known in the art). DNA molecules suitable fordetection with a detectable oligonucleotide probe or primer/templateoligonucleotide of the invention include, but are not limited to, doublestranded DNA (e.g., genomic DNA, plasmid DNA, mitochondrial DNA, viralDNA, and synthetic double stranded DNA). Single-stranded DNA targetnucleic acid molecules include, for example, viral DNA, cDNA, andsynthetic single-stranded DNA, or other types of DNA known in the art.

In general, a target sequence for detection is between 10 and 100nucleotides in length (e.g., 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70,80, 90, 100 nucleotides. The GC content of the target nucleic acidmolecule is selected to be less than about 45, 50, 55, or 60%.Desirably, the target sequence and nicking enzymes are selected suchthat the target sequence does not contain nicking sites for any nickingenzymes that will be included in the reaction mix.

Detectable Oligonucleotide Probes

The present invention provides for the quantitative detection of targetnucleic acid molecules or amplicons thereof in a NEAR reaction usingnon-amplifiable detectable polynucleotide probes comprising at least onepolymerase-arresting molecule (e.g., nucleotide modification or othermoiety that renders the oligonucleotide capable of binding a targetnucleic acid molecule, but incapable of supporting template extensionutilizing the detectable oligonucleotide probe as a target). Withoutwishing to be bound by theory, the presence of one or more moietieswhich does not allow polymerase progression likely causes polymerasearrest in non-nucleic acid backbone additions to the oligonucleotide orthrough stalling of a replicative polymerase (i.e. C3-spacer, damagedDNA bases, other spacer moiety, O-2-Me bases). These constructs thusprevent or reduce illegitimate amplification of the probe during thecourse of a NEAR reaction. This distinguishes them from conventionaldetection probes, which must be added at the end of the NEAR reaction toprevent their amplification.

Conventional detection probes have proven impractical for quantitating aNEAR reaction in real time. If conventional detection probes areincorporated into the NEAR reaction, these conventional detection probesare amplified concurrently with the target. The amplification of thesedetection molecules masks the detection of legitimate target ampliconsdue to the number of starting molecules of the detection probe at thestart of the reaction.

The invention provides non-amplifiable detectable polynucleotide probethat comprise least one polymerase-arresting molecule. Apolymerase-arresting molecule of the invention includes, but is notlimited to, a nucleotide modification or other moiety that blocksprimer-template extension by replicative DNA polymerases, therebypreventing the amplification of detection molecules; but can allowproper hybridization or nucleotide spacing to the target molecule oramplified copies of the target molecule. In one embodiment, a detectableoligonucleotide probe of the invention comprises a 3 carbon spacer(C3-spacer) that prevents or reduces the illegitimate amplification of adetection molecule.

In one embodiment, the detectable oligonucleotide probe of the inventionis a hair-pin shaped oligonucleotide comprising a detectable moiety. Inanother embodiment, the non-amplifiable detectable polynucleotide probeis a hair-pin shaped oligonucleotide that comprises a fluorophore on oneend and a quenching dye on the opposite end. The loop of the hair-pincomprises a sequence that is complementary to and capable of hybridizingwith a target sequence. The stem of the hair-pin is formed by annealingof complementary arm sequences located on either side of the loop. Afluorophore and a quenching molecule are covalently linked at oppositeends of each arm. When the detectable oligonucleotide probe is in thehair pin configuration, the fluorescent and quenching molecules areproximal to one another, thereby leading to fluorescence resonanceenergy transfer (FRET) and quenching of the fluorescence of thefluorophore. When the detectable oligonucleotide probe encounters atarget molecule, hybridization occurs; the loop structure is convertedto a duplex conformation with the target molecule, causing separation ofthe fluorophore and quencher molecules resulting in fluorescence (Tyagiet al. Nature Biotechnology 14: March 1996, 303-308).

The detectable oligonucleotide probes are specific to the targetsequence. In one embodiment, a detectable oligonucleotide probecomprises one or more modified nucleotide bases having enhanced bindingaffinity to a complementary nucleotide. Examples of bases include, butare not limited to locked nucleic acids (LNA), 2′ Fluoro amidites, and2′OMe RNA amidites (also functioning as a polymerase arrestingmolecule). Detectable oligonucleotide probes of the invention can besynthesized with different colored fluorophores and may be designed tohybridize with virtually any target sequence. In view of theirremarkable specificity, a non-amplifiable detectable polynucleotideprobe of the invention is used to detect a single target nucleic acidmolecule in a sample, or is used in combination with detectableoligonucleotide probes each of which binds a different target nucleicacid molecule. Accordingly, the non-amplifiable detectablepolynucleotide probes of the invention may be used to detect one or moretarget nucleic acid molecules in the same reaction, allowing thesetargets to be quantitated simultaneously. The present inventionencompasses the use of such fluorophores in conjunction with thedetectable oligonucleotide probes described herein.

Use of Non-Amplifiable Detectable Polynucleotide Probes

Non-amplifiable detectable polynucleotide probe are useful in methodsfor quantitating a target nucleic acid molecule in a nicking andextension amplification reaction (NEAR). The method involves contactinga target nucleic acid molecule under substantially isothermal conditionswith a polymerase, two primer/template oligonucleotides, each of whichspecifically binds to a complementary sequence on the target nucleotidemolecule, a nicking enzyme, and the detectable oligonucleotide probe inthe presence of a suitable buffer and dNTPs, generating ampliconscomprising at least a portion of said target nucleic acid molecule; anddetermining the level of target nucleic acid molecule present in thereaction by quantitating the oligonucleotide probe that hybridizes tothe target nucleic acid molecule in real time during the reaction basedon fluorescent intensity from the probe molecules in the reaction.Advantageously, such methods are useful for monitoring NEAR in realtime.

In general, non-amplifiable detectable polynucleotide probes of theinvention are included in a NEAR reaction that comprises (1) a targetnucleic acid molecule; (2) two template oligonucleotide moleculescomprising some number of oligonucleotides that are complementary to thetarget nucleic acid molecule and a site that can be cleaved by a nickingenzyme; (3) dNTPs; (4) a strand displacing polymerase; and (5) a nickingenzyme. Accordingly, the invention provides a method of using thesecomponents to quantitate a target nucleic acid molecule.

NEAR Assays

The invention provides for the detection of target nucleic acidmolecules amplified in a NEAR assay. Such assays are known in the artand described herein. See, for example, US Patent ApplicationPublication 2009/0081670, PCT Application 2009/012246, and U.S. Pat.Nos. 7,112,423 and 7,282,328, each of which is incorporated herein inits entirety. Polymerases useful in the methods described herein arecapable of catalyzing the incorporation of nucleotides to extend a 3′hydroxyl terminus of an oligonucleotide (e.g., a primer/templateoligonucleotide or other primer) bound to a target nucleic acidmolecule. Such polymerases include those that are thermophilic and/orthose capable of strand displacement. Polymerases useful in methodsdescribed herein lack a 5′-3′ exonuclease activity, which wouldotherwise degrade the displaced single stranded nucleic acid strand.olymerase also has reverse transcriptase activity (e.g., derivatives ofBst (large fragment) DNA polymerase, Therminator DNA polymerase,Therminator II DNA polymerase). Exemplary polymerases include, but arenot limited to the Bst large fragments of Bst DNA polymerase I, E. coliDNA polymerase I (Klenow fragment), Klenow fragment (3′-5′ exo-), T4 DNApolymerase, T7 DNA polymerase, Deep VentR. (exo-) DNA Polymerase, DeepVentR DNA Polymerase, Therminator, Therminator II DNA Polymerase,AmpliTherm DNA Polymerase, SP6 DNA polymerase. The followingnon-limiting examples of Reverse Transcriptases (RT) can be used in thereactions of the present method to improve performance when detecting anRNA sequence: OmniScript (Qiagen), SensiScript (Qiagen), MonsterScript(Epicentre), Transcriptor (Roche), HIV RT (Ambion), SuperScript III(Invitrogen), ThermoScript (Invitrogen), Thermo-X (Invitrogen), ImPromII (Promega).

A nicking enzyme binds to a recognition sequence in double-stranded DNAand cleaves one strand of a double-stranded helix. Nicking enzymes maycleave either upstream or downstream of their recognition site or withinthe enzyme's recognition site. For methods disclosed herein, onlynicking enzymes that cleave the top strand downstream of the recognitionsite can be used to launch repetitive cycles of substrate DNA nickingand nick extension by the polymerase to drive exponential amplificationof the target nucleic fragment between the primer-templates. Ideally,the nicking enzyme is functional under the same reaction conditions asthe polymerase. In a preferred embodiment of the invention, the nickingenzyme is thermostable and active between 50° C. and 60° C. Exemplarynicking enzymes useful for methods disclosed herein include, but are notlimited to, Nt.BspQI(NEB), Nt.BspD6I, Nt.BsmAI(NEB), Nt.AlwI(NEB),Nt.BbvCI(NEB), N.Bst9I(Sibenzyme), and Nt.BstNBI(NEB).

A NEAR reaction typically comprises nucleotides, such as, for example,dideoxyribonucleoside triphosphates (dNTPs). The reaction may also becarried out in the presence of dNTPs that comprise a detectable moietyincluding but not limited to a radiolabel (e.g., ³²P, ³³P, ¹²⁵I, ³⁵S) anenzyme (e.g., alkaline phosphatase), a fluorescent label (e.g.,fluorescein isothiocyanate (FITC)), biotin, avidin, digoxigenin,antigens, haptens, or fluorochromes. The NEAR reaction further comprisescertain salts and buffers that provide for the activity of the nickingenzyme and polymerase.

Advantageously, the NEAR reaction is carried out under substantiallyisothermal conditions where the temperature of the reaction is more orless constant during the course of the amplification reaction. Becausethe temperature does not need to be cycled between an upper temperatureand a lower temperature, the NEAR reaction can be carried out underconditions where it would be difficult to carry out conventional PCR.Typically, the reaction is carried out at about between 35° C. and 90°C. (e.g., 35, 37, 42, 60, 65, 70, 75, 80, or 85° C.). Advantageously, itis not essential that the temperature be maintained with a great degreeof precision. Some variability in temperature is acceptable.

Melt temperature (Tm) and reaction rate modifiers may also be used tolower the melting temperature of the oligonucleotides, such as (but notlimited to) ethylene glycol and glycerol. In addition, DNA polymerasereaction rate modifiers (such as dNTP and magnesium concentration) maybe used to alter the reaction rate to lead to a greater quantificationprecision.

This invention provides methods of monitoring a NEAR reaction in realtime, utilizing NEAR amplification strategy as described above and inU.S. Pat. No. 7,112,423B2 and US20090017452A1. In one embodiment,quantitative NEAR utilizes target nucleic acids amplification alongsidea control amplification of known quantity. The amount of target nucleicacid can be calculated as an absolute quantification or a relativequantification (semi-quantitative) based on the source of the control(exogenous or endogenous control).

Quantitation of the unknown nucleotide sequence can be achieved eitherthrough comparison of logarithmic threshold amplification of the unknownto a series of known target sequences in either a separate set ofreactions or in the same reaction; or as an internal endogenous orexogenous co-amplification product which produces a threshold value,indicative of either a positive result (if the unknown exceeds thethreshold) or negative result (if the unknown does not exceed thethreshold).

Applications

The present invention provides for the real-time monitoring of theisothermal amplification NEAR reaction which can provide a quantitativemeasure of the amount of the starting target nucleic acid. Compositionsand methods of the invention are useful in human diagnostics, where arapid quantitative answer is desired (e.g., detectable amplification inunder 15, 10, 9, 8, 7, 6, 5 min. or less). In particular embodiments,the invention provides for the use of NEAR reaction assays in humandiagnostics in clinical settings. In other embodiments, the inventionprovides for the use of NEAR reaction assays in diagnostic field work,where access to thermocycling equipment is unavailable or would beprohibitively expensive. In still other embodiments, the inventionprovides for the use of NEAR reaction assays in an academic settingwhere rapid quantitative answers are desired.

Kits

The invention also provides kits for the amplification of a targetnucleic acid molecule. Such kits are useful for the detection orquantitation of a target nucleic acid in a biological sample obtainedfrom a subject. Kits of the present invention may comprise, for example,one or more polymerases, forward and reverse primer-templates, and oneor more nicking enzymes, as described herein. Where one target is to beamplified, one or two nicking enzymes may be included in the kit. Wheremultiple target sequences are to be amplified, and the primer-templatesdesigned for those target sequences comprise the nicking enzyme sitesfor the same nicking enzyme, then one or two nicking enzymes may beincluded. Where the primer-templates are recognized by different nickingenzymes, more nicking enzymes may be included in the kit, such as, forexample, 3 or more.

In one aspect, the invention provides a kit for nucleic acidamplification comprising a DNA polymerase; a primary primer-template, asecondary primer-template, a nicking enzyme with specificity to anicking enzyme recognition site within the primer-templates, anddeoxynucleotide triphosphates (dNTP's) (e.g., in a buffered solutioncontaining components sufficient for amplification). In variousembodiments, the primary primer-template and secondary primer-template,each have a 3′-end specific recognition region sequence complementary orsubstantially complementary to the target sequence, where the endspecific recognition region comprises one or more 2′ modifiednucleotides; a 5′-end tail sequence containing a nicking enzymerecognition site upstream of the 3′-end specific recognition regionsequences; and a stabilizing sequence upstream (5′) of the nickingenzyme binding site.

In one aspect, the kits of the present invention comprise a homogenousmix of all NEAR reaction components, including, but not limited to,dNTP's, forward and reverse primer-templates, nicking enzyme,polymerase, a detectable target specific polynucleotide probe, reactionbuffer and stabilizers, except the target nucleic acid.

The kits of the present invention may also comprise one or more of thecomponents in any number of separate containers, packets, tubes (e.g.,<0.2 ml, 0.2 ml, 0.6 ml, 1.5 ml, 5.0 ml, >5.0 ml), vials, microtiterplates (e.g., <96-well, 96-well, 384-well, 1536-well, >1536-well),ArrayTape, and the like, or the components may be combined in variouscombinations in such containers. In various embodiments, the kit furthercomprises a pair of primer-template oligonucleotides capable of bindingto and amplifying a reference sequence. In yet other embodiments, thekit comprises a sterile container which contains the primer-templateoligonucleotides; such containers can be boxes, ampules, bottles, vials,tubes, bags, pouches, blister-packs, or other suitable container formknown in the art. Such containers can be made of plastic, glass,laminated paper, metal foil, or other materials suitable for holdingnucleic acids.

The components of the kit may, for example, be present in one or morecontainers, for example, all of the components may be in one container,or, for example, the enzymes may be in a separate container from thetemplates. The components may, for example, be dried (e.g., dryresidue), lyophilized (e.g., dry cake) or in a stable buffer (e.g.,chemically stabilized, thermally stabilized). Dry components may, forexample, be prepared by lyophilization, vacuum and centrifugal assisteddrying and/or ambient drying. In various embodiments, the polymerase andnicking enzymes are in lyophilized form in a single container, and thetemplates are either lyophilized, freeze dried, or in buffer, in adifferent container. In some embodiments, the polymerase, nickingenzymes, and the templates are, in lyophilized form, in a singlecontainer. In other embodiments, the polymerase and the nicking enzymemay be separated into different containers.

Kits may further comprise, for example, dNTPs used in the reaction, ormodified nucleotides, cuvettes or other containers used for thereaction, or a vial of water or buffer for re-hydrating lyophilizedcomponents. The buffer used may, for example, be appropriate for bothpolymerase and nicking enzyme activity.

The kits of the present invention may also comprise instructions forperforming one or more methods described herein and/or a description ofone or more compositions or reagents described herein. Instructionsand/or descriptions may be in printed form and may be included in a kitinsert. A kit also may include a written description of an Internetlocation that provides such instructions or descriptions.

Kits may further comprise reagents used for detection methods (e.g.,real-time or endpoint), such as, for example, hybridization probes orDNA binding dyes. Kits may further comprise reagents used for detectionmethods, such as, for example, reagents used for FRET, lateral flowdevices, dipsticks, fluorescent dye, colloidal gold particles, latexparticles, a molecular beacon, or polystyrene beads. Detectioncomponents may be incorporated into a lateral flow device. The lateralflow device may be used at a point of care.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are well within the purview of the skilled artisan.Such techniques are explained fully in the literature, such as,“Molecular Cloning: A Laboratory Manual”, second edition (Sambrook,1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture”(Freshney, 1987); “Methods in Enzymology” “Handbook of ExperimentalImmunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells”(Miller and Calos, 1987); “Current Protocols in Molecular Biology”(Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994);“Current Protocols in Immunology” (Coligan, 1991). These techniques areapplicable to the production of the polynucleotides and polypeptides ofthe invention, and, as such, may be considered in making and practicingthe invention. Particularly useful techniques for particular embodimentswill be discussed in the sections that follow.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the assay, screening, and therapeutic methods of theinvention, and are not intended to limit the scope of what the inventorsregard as their invention.

EXAMPLES

Currently, the NEAR reaction is used to quickly and isothermally detectthe presence or absence of a target oligonucleotide in a sample. Due totechnical limitations, conventional NEAR methods are unsuitable forquantifying target oligonucleotides in real time due, at least in part,to illegitimate amplification of non-target molecules in the sample,which obscures the detection and accurate quantification of targetamplicons. The present invention provides compositions and methods thatovercome these limitations by providing detectable primer/templates thatare not susceptible to illegitimate amplification. In one embodiment, aquantifiable NEAR assay employs a primer comprising one or more 2′-O-Memodifications that prevents or reduces the illegitimate amplification ofnon-target molecules during the NEAR reaction. Currently, the design ofNEAR amplification assays is limited to very short regions within thetarget nucleic acid that have at least one naturally occurring nickingenzyme recognition site in close proximity. Strand displacementsynthesis initiated from this nick site provides single stranded targetDNA molecules to which primer-templates with short target specificregions can bind and start the cycles of nicking/polymerase extensiontarget amplification reactions. The present invention providescompositions and methods that overcome this limitation by utilizingprimer-templates with longer target specific regions, which thereforeare capable of strand invasion between 50° C. to 60° C. during the firstphase of the amplification reaction without the assistance of stranddisplacement synthesis. Longer target specific regions inprimer-templates come with the disadvantage of providing more realestate to form nonspecific DNA hybrids with extendable 3′-ends that canlaunch synthesis of nonspecific amplification products. Compositions inthe present invention mitigate that disadvantage by extending theplacement of 2′-modified nucleotides beyond a 3′-terminal block of fiveconsecutive modified nucleotides to cover the entire target specificregion utilizing an alternate sequence of 2′-modified and unmodifiednucleotides.

Example 1: Primer-Template Oligonucleotides Comprising 2′-O-MethylNucleotides Reduce or Eliminate Background Signal in NEAR Amplification

When NEAR amplification is performed without input target nucleic acid(i.e., No Target Controls; NTC's) signal is generated despite theabsence of template. Thus, generation of background signal has thepotential to decrease the accuracy of target nucleic acid quantitationusing NEAR amplification. It was hypothesized that the background signalwas generated, in part, by formation of primer-dimers by primer/templateoligonucleotides. Without being bound to theory, polymerase arrestingstructures comprising 2′ modified nucleotides could by used to reduce oreliminate intermolecular and/or intramolecular interactions ofprimers/templates (e.g., primer-dimer formation), and, thereby, reduceor eliminate the background signal in NEAR assay.

Exemplary polymerase-arresting entity structures from 5′ to 3′ comprisea stabilizer sequence, nicking enzyme recognition sequence, nickingenzyme spacer sequence, and target specific recognition sequence, thetarget specific recognition sequence comprising one or more 2′ modifiednucleotides (e.g., 2′-O-methyl ribonucleotides). Where two or more 2′modified nucleotides are present in the target specific recognitionsequence the 2′ modified nucleotides may be contiguous (e.g., 2, 3, 4, 5or more 2′ modified nucleotides). Titration of Clavibacter michiganensissepidonicus (Cms) synthetic double stranded target DNA molecule wasevaluated using detection via a fluorobeacon. Target DNA was seriallydiluted from a stock solution of a synthesized 250 basepair DNA‘longmer’ which was designed with the target sequence and a single nicksite.

The signal in the No Target Controls (NTC's) was suppressed in the2′-O-methyl modified template-containing reactions (FIG. 2A). TheStandard Curve displayed a wide dynamic range using the 2′-O-methyltemplate reactions (FIG. 2B). Samples (10 μl) of the No Target Controlreactions were analyzed via HPLC/Mass Spectrometry and confirmedsuppression of background amplification products (FIG. 2C). The spectraderived from reactions using unmodified oligos showed a complex spectrumcomposed of multiple amplification products derived from non-specificbackground products along with unreacted templates (FIG. 2C, leftpanel). The spectra derived from reactions using 2′-O-methyl modifiedoligos showed a simple spectrum composed of unreacted templates withoutthe presence of non-specific background products (FIG. 2C, right panel).

To study the effect of the 2′-O-methyl modified template-containingreactions on the amplification of a biological sample, genomicClavibacter michiganensis sepidonicus (Cms) was used as target DNA.Amplified products were detection by SYBR green, which detectsdouble-stranded DNA (FIGS. 3A and 3B) or Molecular Beacons, whichdetected a specific product (FIGS. 4A and 4B). Standard reactions werecarried out with DNA oligonucleotide templates (FIGS. 3A and 4A) and2′-O-methyl modified template-containing reactions (DNAble) reactionswere carried out with oligonucleotides containing a block of 5contiguous 2′-O-methyl nucleotides at the 3′-end in the target specificrecognition sequence (FIGS. 3B and 4B). The signal in the No TargetControls (NTC's) was suppressed in the 2′-O-methyl modifiedtemplate-containing reactions (FIGS. 3B and 4B) whereas significantsignal is observed in the No Target Controls (NTC's) (FIGS. 4A and 4B)indicating generation of background product in the absence of targetDNA.

Thus, these results indicate that primers comprising 2′-O-methylnucleotides reduce or eliminate background signal in NEAR amplification.

Example 2: Positioning of 2′-O-Methyl Nucleotides in Primer/TemplateOligonucleotides Altered Time-to-Detection and Efficiency of NEARReactions

Exemplary polymerase arresting entities having 2′-O-methyl modifiednucleotides at different positions within the specificity region wereused in NEAR amplification reactions and their reaction kineticsstudied. In particular, the primer/templates studied included a pair ofoligonucleotides having a block of five 2′-O-methyl nucleotides placedat the 3′ end of the specificity region or at the 5′ end of thespecificity region (2 nucleotides downstream of the nick site) (FIG. 5).Standard reactions were carried out in duplicate with a block of2′-O-methyl nucleotides on the 3′-end or starting at the 3^(rd)nucleotide after the nick site and continuing for 5 bases or a mixtureof these two structures as indicated. Target DNA was genomic Clavibactermichiganensis sepidonicus (Cms). Detection was based on Molecular Beaconat a final concentration of 100 nM.

Reaction rate modifying entities having 2′-O-methyl modified nucleotidesat different positions within the specificity region of theprimer/template oligonucleotide displayed different amplificationkinetics (FIG. 6). The reactions using primer/templates having a blockof five 2′-O-methyl nucleotides at the 3′ end showed decreased time todetection (“Terminal” template; 170 sec) compared to primer/templateshaving a block of five 2′-O-methyl nucleotides starting at the 3^(rd)nucleotide after the nick site (“Nick+2” template”; 430 sec). Thus, itwas hypothesized that ratios of the two primer/template oligos can beused to manipulate the time-to-detection and/or the efficiency of thereaction for the ‘tuning’ of reactions. Reactions with varying ratios of“Terminal” template:“Nick+2” template showed intermediatetime-to-detection between that of the two templates (FIG. 6).Additionally, with increasing ratio of “Terminal” template:“Nick+2”template, the curve was contracted and the slope of the curve shifted.Thus, it was shown that positioning of 2′ modified nucleotides inprimer/template oligonucleotides and ratios of primer/templateoligonucleotides with differentially positioned 2′ modified nucleotidesaltered time-to-detection and efficiency of NEAR reactions. Theinvention is at least based in part on these discoveries.

Example 3: Complete Suppression of Nonspecific Amplification in NEARAssays Utilizing Primer-Templates with Long Target Specific Regions

A NEAR assay for the quantification of the corn alcohol dehydrogenase 1gene (ADH1) was designed utilizing two alternative sets of forward andreverse primer-templates (TS3 & TS3). No suitable nicking enzymerecognition site could be located within 500 nucleotides upstream ordownstream from the target sequence region in corn gDNA. Both sets ofprimers-templates feature longer target complementary regions (16 and 19nucleotides, respectively) capable of strand invasion mediatedhybridization with the target DNA. In the first set (TS3) the targetcomplementary regions of the forward and reverse primer-templatescontain a block of 5 consecutive 2′-O-methyl modified ribonucleotidesimmediately upstream from the 3′-terminal deoxynucleotide. The remainderof the target sequence complementary region comprises a sequence ofalternating unmodified deoxynucleotides and 2′-O-methyl ribonucleotidesstarting five nucleotides (forward primer-template) or four nucleotides(reverse primer-template) downstream from the nick site. The second set(TS6) of primer-templates features only a block of five 2′-O-methylribonucleotides adjacent to the 3′-terminal unmodified deoxynucleotide,while the rest of the target complementary region comprises onlyunmodified dexynucleotides.

Ten microliter NEAR reactions were set up in 50 mM Tris pH 8.0, 15 mM(NH₄)₂SO₄, 15 mM Na₂SO₄, and 15 mM MgSO₄ using 3.84 U Warmstart 2.0 BstDNA polymerase 1 (NEB), 10K copies of synthetic corn ADH1 target DNA,0.3 mM dNTP's, 3 U Nt.BstNBI nicking enzyme, 200 nM ROX/BHQ-labeled ADH1molecular beacon probe, 0.5× SYBRgreen dye (LifeTechnologies), 1000 nMTS3 or TS6 reverse primer-template and 100 nM TS3 or TS6 forwardprimer-template. A set of no target DNA control reactions (NTC) weremade up of the same components without the synthetic corn ADH1 targetDNA. All reactions were incubated at 56° C. for 15 minutes andfluorescence signals recorded at 520 nm (SYBRgreen) and 610 nm (ROX).

Comparing the amplification plots of the target DNA containing reactionsin the SYBRgreen (FIG. 8A) and ROX (FIG. 8B) detection channels withamplification plots of the NTC reactions in the SYBRgreen detectionchannel (FIG. 8C)

Results reported herein were obtained using the following methods andmaterials unless indicated otherwise.

NEAR Amplification Reactions

Reactions (50 μl) contained 15 mM MgSO₄, 0.3 mM dNTPs, 19.2 units BstPolymerase, 15 units n.BstNBI, 1000 nM template 1, and 200 nM template2. Target DNA was genomic Clavibacter michiganensis sepidonicus (Cms) ora “longmer” based on Cms sequences. Templates and target werepreincubated together at 56° C. for 30 seconds in a total volume of 10μl. Master mix of the remaining reaction components was preincubated at56° C. for 30 seconds in a total volume of 40 μl. Master mix wascombined with the templates and target, and incubated at 56° C. for 10minutes with fluorescent detection (SYBR Green or Molecular Beacons)collected every 10 seconds during incubation. Reactions were ‘heatkilled’ with a 2 minute 95° C. step followed by a return to roomtemperature. Cycle threshold (Ct) equivalents were determined for eachreaction based on a curve fit formula in the Biorad IQ5 software andvalues were plotted on a graph using Microsoft Excel. A linearregression was carried out and a Correlation Coefficient (R²) wasdetermined.

Other Embodiments

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

This application may be related to International Patent Application No.PCT/US2011/047049, filed Aug. 9, 2011, which claims the benefit of U.S.Provisional Application No. 61/373,695, filed Aug. 13, 2010, the entirecontents of which are incorporated herein by reference.

All patents and publications mentioned in this specification are hereinincorporated by reference to the same extent as if each independentpatent and publication was specifically and individually indicated to beincorporated by reference.

What is claimed is:
 1. An isolated oligonucleotide comprising from 5′ to3′, i) a first region, and ii) a second region, wherein the first regioncomprises a nicking enzyme recognition sequence; wherein the secondregion comprises at least 9 nucleotides that specifically bind acomplementary sequence on a target nucleic acid molecule; and whereinthe second region comprises one or more 2′ modified nucleotidespositioned at the 3′ terminus of the sequence complementary to thetarget nucleic acid molecule.
 2. The isolated oligonucleotide of claim1, wherein the 2′ modification is selected from the group consisting of2′-O-methyl, 2′-methoxyethoxy, 2′-fluoro, 2′-hydroxyl, 2′-allyl,2′-O-[2-(methylamino)-2-oxoethyl], 4′-thio, 4′-CH₂—O-2′-bridge, 4′-(CH₂)2-O-2′-bridge, 2′-LNA, and 2′-O-(N-methylcarbamate) and base analogs. 3.The isolated oligonucleotide of claim 1, wherein two or more 2′ modifiednucleotides are contiguous.
 4. The isolated oligonucleotide of claim 3,wherein the number of contiguous 2′ modified nucleotides is 2, 3, 4, 5,or more.
 5. The isolated oligonucleotide of claim 4, wherein 5contiguous 2′-O-methyl modified nucleotides are positioned at the 3′ endof the sequence complementary to the target nucleic acid molecule. 6.The isolated oligonucleotide of claim 1, wherein the nicking enzymerecognition sequence is 5′-GAGT-3′.
 7. An isolated oligonucleotidecomprising from 5′ to 3′, i) a first region, and ii) a second region,wherein the first region comprises a nicking enzyme recognitionsequence; wherein the second region comprises at least 9 nucleotidesthat specifically bind a complementary sequence on a target nucleic acidmolecule; and wherein the second region comprises one or more 2′modified nucleotides positioned at the 5′ terminus of the sequencecomplementary to the target nucleic acid molecule.
 8. The isolatedoligonucleotide of claim 7, wherein the 2′ modification is selected fromthe group consisting of 2′-O-methyl, 2′-methoxyethoxy, 2′-fluoro,2′-hydroxyl, 2′-allyl, 2′-O-[2-(methylamino)-2-oxoethyl], 4′-thio,4′-CH₂—O-2′-bridge, 4′-(CH₂)₂-0-2′-bridge, 2′-LNA, and2′-O-(N-methylcarbamate) and base analogs.
 9. The isolatedoligonucleotide of claim 7, wherein the one or more 2′ modifiednucleotides positioned at the 5′ end of the sequence complementary tothe target nucleic acid molecule are separated from the nick site by 1,2, 3, 4, 5 or more unmodified nucleotides.
 10. The isolatedoligonucleotide of claim 7, wherein two or more 2′ modified nucleotidesare contiguous.
 11. The isolated oligonucleotide of claim 10, whereinthe number of contiguous 2′ modified nucleotides is 2, 3, 4, 5, or more.12. The isolated oligonucleotide of claim 11, wherein 5 contiguous2′-O-methyl modified nucleotides are positioned at the 5′ end of thesequence complementary to the target nucleic acid molecule.
 13. Theisolated oligonucleotide of claim 7, wherein the nicking enzymerecognition sequence is 5′-GAGT-3′.